Nitroaniline derivatives and their use as anti-tumour agents

ABSTRACT

The invention provides nitroaniline derivatives represented by general formula (1) where the nitro group is substituted at any one of the available benzene positions 2-6; where R and A separately represent the groups NO 2 , CN,COOR 1 , CONR 1  R 2 , CSNR 1  R 2  or SO 2  NR 1  R 2  and A is substituted at any one of the available benzene positions 2-6; where B represents N(CH 2  CH 2  halogen) 2  or N(CH 2  CH 2  OSO 2  R 3 ) 2  substituted at any one of the available benzene positions; and where R 1 , R 2  and R 3  separately represent H, or lower alkyl optionally substituted with hydroxyl, ether, carboxy or amino functions, including cyclic structures, or R 1  and R 2  together with the nitrogen form a heterocyclic structure, and pharmaceutical preparations containing them. These compounds have activity as hypoxia-selective cytotoxins, reductively-activated prodrugs for cytotoxins, hypoxic cell radiosensitisers, and anticancer agents. ##STR1##

This is a division of application Ser. No. 08/244,449, filed asPCT/GB92/02199 Nov. 27, 1992, U.S. Pat. No. 5,571,845.

Alkylating agents are an important class of anticancer drugs, whichexpress their cytotoxic and antitumour effects by forming adducts withcellular DNA (Garcia et al., Biochem. Phannacol, 1988, 37, 3189).

The present invention relates to novel nitroaniline-based alkylatingagents having activity as hypoxia-selective cytotoxins,reductively-activated prodrugs for cytotoxins, hypoxic cellradiosensitisers, and anticancer agents to methods of preparing thenovel compounds, and to the use of these compounds as antitumour agents.

In one aspect, the present invention relates to the class ofnitroaniline derivatives represented bv the general formula (I);##STR2## where the nitro group is substituted at any one of theavailable benzene positions 2-6; where R and A separately represent thegroups NO₂, CN, COOR¹, CONR¹ R². CSNR¹ R² or SO₂ NR¹ R² and A issubstituted at one of the available benzene positions 2-6; where Brepresents N(CH₂ CH₂ halogen)₂ or N(CH₂ CH₂ OSO₂ R³)₂ substituted at anyone of the available benzene positions; and where R¹, R² and R³separately represent H, or lower alkyl optionally substituted withhydroxyl, ether, carboxyl or amino functions, including cyclicstructures, or R¹ and R² together with the nitrogen form a heterocyclicstructure.

Where R¹, R² and/or R³ represent lower alkyl the group may contain from1 to 6 carbon atoms.

Where R¹, R² and/or R³ represent groups containing tertiary amines, theN-oxides of those tertiary amine moieties are also included.

The compounds of formula (I) have cytotoxic and antitumour activity andare useful as hypoxia-selective cytotoxins, reductively-activatedprodrugs for cytotoxins. hypoxic cell radiosensitisers, and anticanceragents.

Some of the compounds of formula (I) form pharmaceutically-acceptableaddition salts with both organic and inorganic acids, and these additionsalts also form part of the present invention. Examples of suitableacids for salt formation are hydrochloric, sulfuric, phosphoric, acetic,citric, oxalic, malonic, salicylic, malic. fumaric, succinic, ascorbic,maleic, methanesulfonic, isethionic and the like. Some of the compoundsof formula (I) form pharmaceutically-acceptable addition salts with bothorganic and inorganic bases, and these addition salts also form part ofthe present invention. Examples of suitable bases for salt formation aresodium and potassium carbonate, sodium and potassium hydroxide, ammonia,triethylamine. triethanolamine and the like.

The compounds of formula (I) and the acid or base addition salts and the1N-oxides thereof may be prepared by the processes outlined in thefollowing Schemes 1 to 7.

Specific but non-limitative examples of the processes outlined inSchemes 1 to 7 are given hereinafter in Examples A to H respectively.##STR3##

The invention also relates to the preparation of compounds of thegeneral formula I, the acid or base addition salts and the N-oxidesthereof, by a process as outlined in any one of Schemes 1-7 above or byan obvious chemical equivalent thereof. The invention includes alsothose forms of the preparation processes according to which a compoundobtainable as intermediate at any stage of the process is used asstarting material and the remaining steps are carried out, or a startingmaterial is used in the form of a derivative or salt or, especially, isformed under the reaction conditions.

The following Table 1 sets out physicochemical data for 24 compoundswithin the general formula (I), representative of it, and preparable bythe processes of the invention.

                                      TABLE 1                                     __________________________________________________________________________    Analytical and physicochemical data for nitroaniline mustards of general      formula 1.                                                                    No.                                                                              A   NO.sub.2                                                                         B         R           mp (°C.)                                                                     formula     analyses.sup.a              __________________________________________________________________________     1 2-NO.sub.2                                                                        4  5-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         CONH.sub.2  109-111                                                                             C.sub.11 H.sub.12 Cl.sub.2 N.sub.4                                            O.sub.5     C, H, N, Cl                  2 2-NO.sub.2                                                                        4  5-N(CH.sub.2 CH.sub.2 Br).sub.2                                                         CONH.sub.2  126-128                                                                             C.sub.11 H.sub.12 Br.sub.2 N.sub.4                                            O.sub.5     C, H, N                      3 2-NO.sub.2                                                                        4  5-N(CH.sub.2 CH.sub.2 I).sub.2                                                          CONH.sub.2  170-172                                                                             C.sub.11 H.sub.12 I.sub.2 N.sub.4                                             O.sub.5     C, H, N, I                   4 2-NO.sub.2                                                                        4  5-N(CH.sub.2 CH.sub.2 OMs).sub.2.sup.b                                                  CONH.sub.2  142-144                                                                             C.sub.13 H.sub.18 N.sub.4 O.sub.11                                            S.sub.2     C, H, N, S                   5 2-NO.sub.2                                                                        4  5-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         COOMe       136.5-138                                                                           C.sub.12 H.sub.13 Cl.sub.2 N.sub.3                                            O.sub.6     C, H, N, Cl                  6 2-NO.sub.2                                                                        4  5-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         COOH        125-127                                                                             C.sub.11 H.sub.11 Cl.sub.2 N.sub.3                                            O.sub.6.1/2EtOAc                                                                          C, H, N                      7 2-NO.sub.2                                                                        4  5-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         CN          127   C.sub.11 H.sub.10 Cl.sub.2 N.sub.4                                            O.sub.4     C, H, N                      8 2-NO.sub.2                                                                        4  5-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         CONHMe      180.5 C.sub.12 H.sub.14 Cl.sub.2 N.sub.4                                            O.sub.4     C, H, N, Cl                  9 2-NO.sub.2                                                                        4  5-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         CONMe.sub.2 130.5 C.sub.13 H.sub.16 Cl.sub.2 N.sub.4                                            O.sub.5     C, H, N                     10 2-NO.sub.2                                                                        4  5-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         CONX.sup.c  140-142                                                                             C.sub.15 H.sub.18 Cl.sub.2 N.sub.4                                            O.sub.6     C, H, N                     11 2-NO.sub.2                                                                        4  5-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         CONH(CH.sub.2).sub.2 NMe.sub.2.HCl                                                        85-90 C.sub.15 H.sub.21 Cl.sub.2 N.sub.5                                            O.sub.5.HCl C, H, N                     12 2-NO.sub.2                                                                        4  5-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         CONH(CH.sub.2).sub.2 NX.sup.c.HCl                                                         119-120                                                                             C.sub.17 H.sub.23 Cl.sub.2 N.sub.5                                            O.sub.6.HCl C, H, N, Cl                 13 2-NO.sub.2                                                                        4  5-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         CSNH.sub.2  166-167                                                                             C.sub.11 H.sub.12 Cl.sub.2 N.sub.4                                            O.sub.4 S   C, H, N, Cl                 14 2-NO.sub.2                                                                        4  5-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         CONH(CH.sub.2).sub.2 N(O)Me.sub.2                                                         165-169                                                                             C.sub.15 H.sub.21 Cl.sub.2 N.sub.5                                            O.sub.6.HCl C, H, N, Cl                 15 2-NO.sub.2                                                                        4  5-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         CONH(CH.sub.2).sub.2 COOH                                                                 150-153                                                                             C.sub.14 H.sub.16 Cl.sub.2 N.sub.4                                            O.sub.7     C, H, N                     16 2-NO.sub.2                                                                        4  5-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         CONHCH.sub.2 CH.sub.2 OH                                                                  157-158                                                                             C.sub.13 H.sub.16 Cl.sub.2 N.sub.4                                            O.sub.6     C, H, N, Cl                 17 2-NO.sub.2                                                                        4  5-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         CONHCH.sub.2 CH(OH)CH.sub.2 OH                                                            gum   C.sub.14 H.sub.18 Cl.sub.2 N.sub.4                                            O.sub.7     C, H, N, Cl                 18 2-NO.sub.2                                                                        4  5-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         SO.sub.2 NH.sub.2                                                                         153-154                                                                             C.sub.10 H.sub.12 Cl.sub.2 N.sub.4                                            O.sub.6 S   C, H, N, S                  19 3-NO.sub.2                                                                        5  5-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         CONH.sub.2  123-124                                                                             C.sub.11 H.sub.12 Cl.sub.2 N.sub.4                                            O.sub.5     C, H, N, Cl                 20 3-NO.sub.2                                                                        5  5-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         CONH(CH.sub.2).sub.2 NMe.sub.2                                                            115-118                                                                             C.sub.15 H.sub.21 Cl.sub.2 N.sub.5                                            O.sub.5     C, H, N, Cl                 21 2-NO.sub.2                                                                        6  5-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         CONH.sub.2    141-141.5                                                                         C.sub.11 H.sub.12 Cl.sub.2 N.sub.4                                            O.sub.5     C, H, N, Cl                 22 2-NO.sub.2                                                                        6  5-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         CONH(CH.sub.2).sub.2 NMe.sub.2.HCl                                                        180-182                                                                             C.sub.15 H.sub.21 Cl.sub.2 N.sub.5                                            O.sub.5.HCl C, H, N, Cl                 23 3-NO.sub.2                                                                        5  4-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         CONH.sub.2  113-116                                                                             C.sub.11 H.sub.12 Cl.sub.2 N.sub.4                                            O.sub.5     C, H, N, Cl                 24 3-NO.sub.2                                                                        5  4-N(CH.sub.2 CH.sub.2 Cl).sub.2                                                         CONH(CH.sub.2).sub.2 NMe.sub.2.HCl                                                        145-148                                                                             C.sub.15 H.sub.21 Cl.sub.2 N.sub.5                                            O.sub.5.HCl.H.sub.2 O                                                                     C, H,                       __________________________________________________________________________                                                      N                            Footnotes for Table 1                                                         .sup.a Analyses for all listed elements within ± 0.4%.                     .sup.b OMs = OSO.sub.2 CH.sub.3.                                              .sup.c X = morpholide: (CH.sub.2 CH.sub.2).sub.2 O.                      

The following Examples A to H illustrate the preparation of compoundsand representative of the general formula (I), and specifically thecompounds listed in Table 1.

EXAMPLE A Preparation of 5-N,N'-bis(2-methanesulfonoxvethyl)amino!-2,4--dinitrobenzamide (4) (Ia:X=OSO₂ Me; R=CONH₂) and analogues by the method outlined in Scheme 1.

Treatment of 5-chloro-2,4-dinitrobenzoic acid (II: Y=OH) Goldstein, H.;Stamm, R. Helv. Chim. Acta, 1952,35, 133014 1332! with SOCl₂ followed byammonia gave 5-chloro-2,4-dinitrobenzamide (II: Y=NH₂), mp 210 ° C.(dec) Goldstein, H.; Starmm, R. Helv. Chim. Acta. 1952, 35, 1330-1332report mp 212° C.!. ¹ H NMR (CD₃ COCD₃) δ8.68 (s, 1 H, H-3), 8.02 (s, 1H, H-6), 7.50 (br, 2 H, CONH₂). This amide was heated withdiethanolamine in dioxan to give 5-N,N-bis(2-hydroxyethyl)!amino-2,4-dinitrobenzanude (III) (45% yield), mp(EtOAc) 176°-178° C. ¹ H NMR (CD₃ SOCD₃) δ8.47 (s, 1 H, ArH3), 8.07,7.73 (2 br, 2 H, CONH₂), 7.35 (s, 1H, ArH6), 4.80 (m, 2H, OH), 3.83-3.20(m, 8 H, CH₂ N, CH₂ O). Anal. (C₁₁ H₁₄ N₄ O₇) C,H,N.

Methanesulfonyl chloride (5.17 ml, 0.067 mol) was added dropwise to asolution of the above amide diol (10.00 g, 0.032 mol) in dry pyridine(150 mL.) at 0° C. After a further 10 minutes at 0° C., the solution wasstirred at 20° C. for 30 minutes, and then volatiles were removed underreduced pressure at below 40° C. The residue was partitioned betweenEtOAc and water, and the organic residue was chromatographed on silicagel. Elution with EtOAc gave foreruns, followed by 5-N,N-bis(2-methanesulfonoxyethyl)amino!-2,4-dinitrobenzamide (4) (Ia:X=OSO₂ Me; R=CONH₂) (11.05 g, 74%), mp (sealed tube: ex EtOAc/petroleumether) 60° C. ¹ H NMR(CD₃ COCD₃) δ8.54 (s, 1 H, H-3), 7.65 (s, 1 H,H-6), 4.49 (t, J=5.3 Hz, 4 H, CH₂ OSO₂ CH₃), 3.89 (t, J=5.3 Hz, 4 H, CH₂N), 3.09(s, 6H, SO₂ CH₃), 2.90(br s, NH₂). ¹³ C NMR δ167.01 (CONH₂),148.56 (C-5), 140.45, 138.44 (C-2,4), 138.57 (C-1), 124.92, 123.06(C-3.6), 67.65 (CH₂ OSO₂ CH₃), 52.02 (CH₂ N), 37.23 (SO₂ CH₃). Anal.(C₁₃ H₁₈ N₄ O₁₁ S₂) C,H,N,S.

The above dimesylate (4) (15.0 g, 31.9 mol) was dissolved in dry DMF(150 mL) The solution was treated with solid LiCl (20 g), warmed to 100°C. with vigorous stirring for 1h, and then concentrated to dryness. Theresidue was partitioned between EtOAc and water, and the organic portionwas washed well with 3N HCl, worked up to give a waxy solid, andchromatographed on silica. Elution with EtOAc/petroleum ether (1:1) gaveforeruns, while EtOAc/petroleum ether (3:2) gave 5-N,N-bis(2-chloroethyl)amino!-2,4-diritrobenzamide (1) (Ia, X=Cl, R=CONH₂) (7.51 g, 67%)which crystallised from EtOAc/petroleum ether assmall orange needles, m.p. 109°-111° C., ¹ H NMR (CD₃ COCD₃)δ8.52 (s,1H, H-3), 758 (s, 1H, H-6), 7.10 (br, 2H, CONH₂), 3.89-3.81 (m, 8H,NCH₂,CH₂,Cl). ¹³ C NMR δ167.10 (CONH₂), 148.31 (C-5), 140.08 (C-2),138.84 (C-1), 138.20 (C-4), 124.94. 122.24 (C-3,6), 54.25 (CH₂,N), 42.04(CH₂ Cl) ^(M) /z 354,352,350 (M+, 2%), 303.301(67,24%),222(25),185(26),171(29),100(74),65(25),63(56),56(57),36(100).FoundM+354.0124; 352.0123; 350.0189. Anal. (C₁₁ H₁₂ Cl₂ N₄ O₅) C, H, N, I.

A solution of the above dimesylate (4) (1.00 g, 2.12 mmol) in dry DMF(50 mL) was treated with solid NaBr (10.0 g), warmed to 120° C. withvigorous stirring for 15 min, and then concentrated to. dryness underreduced pressure. The residue was partitioned between EtOAc and water,and the organic portion was washed well with 3N HCl and worked up.Chromatogaraphy of the residue on silica and elution withEtOAc/petroleum ether (1:1) gave 5-N,N-bis(2-bromoethyl)amino!-2,4-dinitrobenzamide (2) (Ia: X=Br, R=CONH₂)(0.73 g, 78% yield), mp (EtOAc/petroleum ether) 126°-128° C. ¹ H NMR(CD₃ COCD₃) δ8.53 (s, 1 H, H-3), 7.58 (s, 1H, H-6), 3.89 (t, J=6.6 Hz, 4H, CH₂ N), 3.73(t,J=6.6 Hz, 4H, CH₂ Br), 2.93 (br, 2H, CONH₂), ¹³ C NMRδ167.20 (CONH₂), 147.89 (C-5), 140.06, 138.26 (C-2,4), 138.75 (C-1),124.95, 122.30 (C-3,6), 54.11 (CH₂ N), 29.8 (CH₂ Br). Anal.(C₁₁ H₁₂ Br₂N₄,O₅) C,H,N.

A similar reaction using NaI gave 5-N,N-bis(2-iodoethyl)amino!-2,4-dinitrobenzamide (3) (Ia: X=I, R=CONH₂)in 92% yield. mp (EtOAc/petroleum ether) 170°-172° C. ¹ H NMR (CD₃COCD₃) δ8.52 (s, 1 H. H-3), 7.55 (s, 1H, H-6), 3.84 (t, J=7.2 Hz. 4 H,CH₂ N), 3.50 (t, J=7.2 Hz. 4 CH₂ Br), 2.88 (br, 2H, CONH₂). ¹³ C NMRδ167.09 (CONH₂), 147.17 (C-5), 140.08, 138.33 (C-2,4), 138.74 (C-1),124.94, 122.31 (C-3,6), 54.89 (CH₂ N), 1.72 (CH₂ I). Anal. (C₁₁ H₁₂ I₂N₄ O₅) C,H,N,I.

EXAMPLE B Methyl 5- N,N-bis(2-chloroethyl)amino!-2,4-dinitrobenzoate (5)(VI: X=Cl) and analogues by the method outlined in Scheme 2.

Methyl 5-chloro-2,4-dinitrobenzoate (IV) (1.24 g, 4.76 mmol) was treatedwith diethanolarnine (1.00 g, 9.52 mmol) in dioxane (50 mL) at 50 ° C.for 3 h, and the residue after workup was chromatographed on silica gel.Elution with EtOAc gave methyl 5-N,N-bis(2-hydroxyethyl)amino!-2,4-dinitrobenzoate (V) (1.52 g. 97%). mp(EtOAc/petroleum ether) 104°-106° C. ¹ H NMR (CDCl₃) δ8.48 (s, 1 H,H-3), 7.45 (s, 1H, H-6), 3.95 (s, 3H, OCH₃), 3.81 (t, J=5.0 Hz, 4 H, CH₂OH), 3.60 (t,J=5.0 Hz, 4H, CH₂ N), 2.20 (br, 2H, OH). ¹³ C NMR δ165.89(COOMe), 147.53 (C-5), 138.67, 136.21 (C-2,4), 133.30 (C-1), 124.22.121.65 (C-3,6), 59.34 (CH₂ OH), 54.59 (CH₂ N), 53.74 (OCH₃). Anal. (C₁₂H₁₅ N₃ O₈) C,H,N.

A solution of this diol (1.50 g, 4.56 mmol) and Et₃ N (1.33 mL, 9.57mmol) in dry CH₂ Cl₂ (60 mL) was treated with MsCl (0.73 mL 9.34 mmol)at 0° C., and poured into saturated aqueous NaHCO₃ after 15 min. Thecrude dimesylate (VI: X=OSO₂ Me) from workup was treated directly withexcess LiCl in DMF at 120° C. for 30 min. The mixture was poured intobrine, extracted with EtOAc, worked up, and chromatographed on silicagel. Elution with EtOAc/petroleum ether gave methyl 5-N,N-bis(2-chloroethyl)amino!-2,4-dinitrobenzoate (5) (VI: X =Cl; Y¹=OMe) (1.34 g, 80%), mp (EtOAc/petroleum ether) 136.5°-138 ° C. ¹ H NMR(CDCl₃) δ8.54 (s, 1H, H-3), 7.34 (s, 1 H, H-6), 3.97 (s, 3 H, OCH₃),3.68 (br s, 8 H, NCH₂ CH₂ Cl). ¹³ C NMR δ165.30 (COOCH₃), 147.22 (C5),139.81, 137.82 (C-2,4), 133.39 (C-1), 124.33, 121.77 (C-3,6), 53.86(OCH₃), 53.78 (CH₂ N), 40.69 (CH₂ Cl). Anal. (C₁₂ H₁₃ Cl₂ N₃ O₆)C,H,N,Cl.

Hydrolysis of (5) with 4N KOH in p-dioxane at 20° C. for 15 min gave aquantitative yield of 5- N,N-bis(2-chloroethyl)amino!-2,4-dinitrobenzoicacid (6) (Ib: X=Cl, R=COOH), mp (EtOAc/petroleum ether) 125°-127° C. ¹ HNMR (CD₃ COCD₃) δ8.56 (s, 1H, H-3), 7.72 (s, 1H, H-6), 3.89-3.86 (br, 8H, NCH₂ CH₂ Cl), 3.78 (br, COOH). ¹³ C NMR δ166.02 (COOH), 148.52 (C-5),140.25, 138.09 (C-2,4), 134.72 (C-1), 124.87, 122.58 (C-3,6), 54.12 (CH₂N), 42.12 (CH₂ CI). Anal. (C₁₁ H₁₁ Cl₂ N₃ O₆ /1/2EtOAc) C,H,N,Cl.

Treatment of (6) with excess SOCl₂ containing a drop of DMF under refluxfor 20 min, followed by concentration to dryness and azeotroping withbenzene, gave crude 5- N,N-bis(2-chloroethyl)amino!-2,4-dinitrobenzoylchloride (VI: X=Cl; Y¹ =Cl). This was dissolved in Me₂ CO (20 ML),cooled to 0° C., treated with excess 40% aqueous methylamine. Theproduct was purified by chromatography on silica gel, elution withEtOAc/petroleum ether (1:1) giving N-methylN,N-bis(2-chloroethyl)amino!-2,4-dinitrobenzamide (8) (Ib: X=Cl,R=CONHMe), mp (EtOAc/petroleum ether) 180.5° C. ¹ H NMR (CD₃ COCD₃)δ8.53 (s, 1 H., ArH3), 7.75 (br, 1 H, CONH), 7.56 (s, 1H, H-6),3.88-3.80 (m, 8 H, N-CHCH₂ CH₂ Cl), 2.90, 2.89 (2s, 3H, CONHMe). ¹³ NMRδ165.89 (CONH), 148.43 (C-5), 140.05, 138.26 (C-2,4), 138.94 (C-1),125.05, 122.45 (C-3,6), 54.20 (CH₂ N), 42.10 (CH₂ Cl), 26.69 (CONHCH₃).Anal. (C₁₂ H₁₄ Cl₂ N₄ O₅) C,H,N,Cl.

Similar treatment of the acid chloride (VI: X=Cl; Y¹ =Cl) with ammoniaor suitable amines gave:

5- N,N-bis(2-chloroethyl)amino!-2,4-dinitrobenzamide (4) (Ib: X=Cl,R=CONH₂) (67% yield), mp (EtOAc/petroleum ether) 109°-111° C. ¹ H NMR(CD₃ COCD₃) δ8.52 (s, 1H, H-3), 7.58 (s, 1 H, H-6), 7.10 (br, 2 H,CONH₂), 3.89-3.81 (m, 8H, NCH₂ CH₂ Cl). ¹³ C NMR δ167.10 (CONH₂), 148.31(C-5), 140.08 (C-2), 138.84 (C-1), 138.20 (C-4), 124.94, 122.24 (C-3,6),54.25 (CH₂ N), 42.04 (CH₂ Cl). ^(M) /z 354, 352, 350 (M³⁰ , 2%), 303,301 (67, 24%), 222 (25), 185 (26), 171(29), 100(74), 65(25), 63(56),56(57), 36(100). Found M⁺⁺ 354.0124; 352.0123; 350.0189. C₁₁ H₁₂ Cl₂ N₄O₅ requires 354.0126; 352.0155; 350.0185. Anal. (C₁₁ H₁₂ Cl₂ N₄ O₅)C,H,N,Cl.

N,N-dimethyl N,N-bis(2-chloroethyl)amino!-2,4-dinitrobenzainide (9) (Ib:X=Cl, R=CONMe₂) (65% yield), mp (CHCl₃ /petroleum ether) 130.5° C. ¹ HNMR (CDCl₃) δ8.61 (s, 1 H, H-3), 7.07 (s, 1 H, H-6), 3.62 (s, 8 H, NCH₂CH₂ Cl), 3.07,2.78 (2×3H, CON(CH₃)₂)). ¹³ C NMR δ166.12 (CON(CH₃)₂)),148.33 (C-5), 138.88, 135.75 (C-2,4), 137.82 (C-1), 124.92, 120.00(C-3,6), 53.55 (CH₂ N), 40.98 (CH₂ Cl), 38.06,34.85 (CON(CH₃)₂)). Anal.(C₁₃ H₁₆ Cl₂ N₄ O₅) C,H,N.

N-morpholino N,N-bis(2-chloroethyl)amino!-2,4-dinitrobenzamiide (10)(Ib: X=Cl, R=CON(CH₂ ; CH₂)₂ O), mp (CHCl₃ /hexane) 140°-142° C. ¹ H NMR(CDCl₃) δ8.66 (s, 1H, ArH-3), 7.55 (s, 1 H, ArH-6), 3.68 (br, 10H,NCH₂,CH₂,Cl and CH₂ O ), 3.60 & 3.34 (2×m, 2×2 H, CH₂ NCO). ¹³ C NMRδ165.22 (CON), 149.34 (C-5), 139.88, 137.00 (C-2,4), 138.26 (C-1),125.59, 121.17 (C-3,6), 66.92, 66.79 (CH₂ O), 54.22 (CH₂ N, 47.88, 42.80(CONHCH₂), 42.26 (CH₂ Cl). Anal. (C₁₅ H₁₈ Cl₂ N₄ 0₆) C,H,N.

N- 2-(dimethylamino)ethyl!N,N-bis(2-chloroethyl)amino!-2,4-dinitrobenzamide (11) (Ib: X=Cl,R=CONHCH₂ CH₂ NMe₂), mp (hydrochloride salt from MeOH/EtOAc) 85°-90° C.¹ H NMR (D₂ O) δ8.76 (s, 1H, ArH-3), 7.59 (s, 1H. ArH-6), 3.86-3.80 (m.10H, NCH₂ CH₂ Cl and CH₂ NH^(+Me) ₂), 3.53 t, J=6.1 Hz, 2H, CONHCH₂),3.06 (s, 6H, NMe₂) ¹³ C NMR δ170.77 (CONH), 166.12, 1451.39 (C-5),140.53, 137.51 (C-2,4), 138.18 (C-1), 128.25, 123.39 (C-3,6), 58.46 (CH₂N⁺ HMe₂), 55.49 (CH₂ N), 45.67 (N⁺ HMe₂), 43.97 (CH₂ Cl), 38.54(CONHCH₂). Anal. (C₁₅ H₂₁ Cl₂ N₅ O₅.HCl) C,H,N.

A solution of the free base of (11) (0.50 g, 1.18 mmol) in CH₂ Cl₂ (15mL) was treated with a solution of2-benzenesulfonyl-3--phenyloxaziridine Davis, F.; Stringer, C.D. J. Org.Chem., 1982, 47, 1775-1777!. (0.32 g, 1.24 mmol) in CH₂ Cl₂ (3 mL) wasadded dropwise to. After 10min, petroleum ether (10 mL) was added, thesolution was cooled at -30° C. overnight, and the resulting solid wasdried under high vacuum to give N-(N¹,N¹ -dimethylaminoethyl)N,N-bis(2-chloroethyl)amino!-2,4-dinitrobenzamide N¹ -oxide (14) ((Ib:X=Cl, R=CONHCH₂,CH₂ N(O)Me₂) as a hygroscopic foam. Treatment withMeOH/HCl gave the hydrochloride salt (0.36g, 64%), mp (MeOH/^(i) Pr₂ O)165°-169° C. ¹ H NMR (D₂ O/(CD₃)₂ SO) δ8.82 (s, 1H, H-3), 7.46 (s, 1H,H-6), 3.98, 3.93 (2xt, J=5.1 Hz, 2×2 H, CH₂ CH₂), 3.82 (t, J=4.7 Hz, 4H,CH₂ Cl), 3.77 (t, J=4.7 Hz, 4 H, CH₂ N), 3.57 (s, 6 H, N(CH₃)₂). ¹³ CNMR (D₂ O/(CD₃)₂ SO) δ170.71 (COHH), 151.63 (C-5), 140.68, 137.31(C-3,6), 69.20 (CH₂ NOMe₂), 59.03 (NOMe₂), 55.40 (CH₂ N), 44.06 (CH₂Cl), 36.45 (CONH CH2). Anal. (C₁₅ H₂₁ Cl₂ N₅ HCl) C,H,N,Cl.

N- 2-(morpholino)ethyl!N,N-bis(2-chloroethyl)amino!-2,4-dinitrobenzainide (12) (Ib:X=Cl,R=CONHCH₂ CH₂ N(CH₂ CH₂)₂ O) as a gum. The hydrochloride saltcrystallized from MeOH/^(i) Pr₂ O, mp 119°-120° C. ¹ H NMR (D₂ O) δ8.81(s, 1 H, H-3), 7.59 (s, 1 H, H-6), 4.07 (br, NH and HCl), 3.91 (t, J=6.0Hz, 4 H, N^(+CH) ₂ CH₂ O), 3.86-3.81 (m, 10 H, NCH₂ CH₂ Cl and CH₂ CH₂N⁺), 3.56 (t, J=6.0 Hz, 6 H, N⁺ CH₂ O and CONHCH₂). ¹³ C NMR δ171.17(CONH), 151.69 (C-5), 140.61, 137.28 (C-2,4), 138.12 (C-1) 128.53,123.48 (C-3,6), 66.30 (N⁺ CH₃ C_(H) 2O),58.07 (CH₂ N⁺),55.48 (CH2N),54.65 (N^(+CH) ₂ CH₂ O), 44.09 (CH₂ Cl), 36.84 (CONHCH₂). Anal. (C₁₇ H₂₃Cl₂ N₅ O₅.HCl.1/2H₂ O) C,H,N,Cl.

N-(2-hydroxyethyl) S- N,N-bis(2-chloroethyl)amino!-2,4-dintrobenzamide(16)(Ib: X=Cl, R=CONHCH₂ CH₂ OH) (1.24 g, 85%), mp (EtOAc/petroleumether) 157°-158° C. ¹ H NMR ((CD₃)₂ SO) δ8.73 t,J=5.5 Hz, 1 H, CONH),8.53 (s, 1 H, H-3), 7.45 (s, 1 H, H-6), 4.76 (t, J=5.5 Hz, 1 H. OH),3.82 (t, J=6.0 Hz, 4 H, CH₂ Cl), 3.68 (t, J=6.0 Hz, 4 H, CH₂ N), 3.54(dt, J=6.1, 5.5 Hz, 2 H, CH₂ OH), 3.31 (dt, J=6.1, 5.5 Hz, 2 H,CONHCH₂). ¹³ C NMR ((CD₃)SO) δ164.44 (CONH), 147.11 (C-5), 137.87.136.27 (C-2,4), 137.32 (C-1), 124.24, 121.01 (C-3,6), 59.32 (CH₂ OH),52.44 (CH₂ N), 41.93 (CONHCH₂), 41.65 (CH₂ Cl). Anal. (C₁₃ H₁₆ Cl₂ N₂O₆) C,H,N,Cl.

N-(2,3-dihydroxypropyl)5-N,N-bis(2-chloroethyl)amino!-2,4-dinitrobenzarnide(17) (Ib: X=Cl,R=CONHCH₂ CH(OH)CH₂ OH) (0.96 g, 59%) as a hygroscopic yellow foam. ¹ HNMR ((CD₃)₂ SO) δ8.70 (t,J 5.7 Hz, 1 H, CONH), 8.53 (s, 1 H, H-3), 7.44(s, 1 H, H-6), 4.83 (d, J=4.9 Hz, 1 H. CHOH) 4.56 (t, J=5.7 Hz, 1 H, CH₂OH), 3.83 (t,J=5.9 Hz, 4 H, CH₂ Cl), 3.68 (t,J=5.9 Hz, 4 H, CH₂ N),3.50-3.06 (m, 5 H, CONH CH₂ CHOHCH₂ OH). ¹³ C NMR ((CD₃)₂ SO) δ164.61(CONH), 147.12 (C-5), 37.94, 136.40 (C-2,4), 137.40 (C-1), 124.29,121.18 (C-3,6). 70.08 (CHOH), 63.77 (CH₂ OH), 52.55 (CH₂ N), 42.79(CONHCH₂), 41.72, (CH₂ Cl). Anal. (C₁₄ H₁₈ Cl₂ N₄ O₇) C,H,N,Cl.

crude N-(2-methoxycarbonylethyl) 5-N,N-bis(2-chloroethyl)amino!-2,4-dinitrobenzarnide from treatment withβ-alanine methyl ester hydrochloride! (Ib: X=Cl, R=CONHCH₂ CH₂ COOMe) asa yellow oil. This was immediately dissolved in THF (25 mL) and treatedwith aqueous KOH (10 mL of 2N) at 20° C. for 3h. The solution was thenacidified with conc. HCl, extracted with EtOAc, and worked up as usual.Chromatography on silica, eluting with EtOAc, gave N-(2-carboxyethyl) 5-N,N-bis(2-chloroethyl)amino!-2,4-dinitrobenzamide (15) (Ib: X=Cl,R=CONHCH₂ CH₂ COOH) (0.72 g, 68%), mp (EtOAc/petroleum ether) 150°-153°C. ¹ H NMR ((CD₃)₂ SO) δ12.27 (br, 1 H, COOH) 8.79 (t,J=5.6 Hz, 1 H,CONH), 8.54 (s, 1 H, H-3), 7.42 (s, 1 H, H-6) 3.83 (t,J=6.0 Hz, 2 H, CH₂Cl), 3.68 (t,J=6.0 Hz, 4 H, CH₂ N), 3.44 (dxt, J=6.9, 2 H, 5.6 Hz,CONHCH₂), 2.53 (t, J=6.9 Hz, 2 H, CH₂, COOH). ¹³ C NMR ((CD₃)₂ SO)δ172.74 (COOH), 164.53 (CONH), 147.21 (C-5), 137.94, 136.13 (C-2,4),137.29 (C-1), 124.37. 120.93 (C-3,6), 52.58 (CH₂ N), 41.72 (CH₂ Cl),35.24, 33.19 (CONHCH₂ CH₂). Anal. (C₁₄ H₁₆ Cl₂ N₄ O₇) C,H,N,Cl.

EXAMPLE C Preparation of5-N,N-bis(2-chloroethyl)aminol!-2,4-dinitrobenzonitrile (7) (Ic: X=Cl,R=CN) and 5- NN-bis(2-chloroethyl)amino!-2,4-dinitrothiobenzamide(13)(Ic: X=Cl, R=CSNH2) by the method outlined in Scheme 3.

A solution of 5- N,N-bis(2-chloroethyl)amino!-2,4-dinitrobenzanide (1)(VII) (0.20 g, 0.57 mmol) in SOCl₂ (5 mL) was heated under reflux in N₂for 84 h. Excess SOCl₂ was removed under reduced pressure, and theresidue was chromatographed on silica gel. Elution with EtOAc/petroleumether (3:7) gave 5-N,N-bis(2-chloroethyl)aminol!-2,4-dinitrobenzonitrile (7)(Ic: X=Cl,R=CN)(0.16 g, 87%), mp (CHCl₃) 127° C. ¹ H NMR (CD₃ COCD₃) δ8.79 (s, 1H, H-3), 8.19 (s, 1 H, H-6),3.93 (s, 8 H, NCH₂ CH₂ Cl). ¹³ C NMR δ148.55(C-5), 140.98, 139.07 (C-2, 4), 129.50, 126.10 (C-3,6), 115.05 (C-1),112.84 (CN), 54.18 (CH₂ N), 42.20 (CH₂ Cl). Anal. (C₁₁ H₁₀ Cl₂ N₄ O₄)C,H,N.

A solution of (1) (VII) (0.50 g, 1.42 mmol) in p-dioxane (20 mL) wastreated with P₂ S₅ (0.63 g, 2.84 mmol) and NaHCO₃ (0.24 g, 2.84 mmol)was heated under reflux with stirring for 3 h. The residue after workupwas chromatographed on silica gel, and elution with EtOAc/petroleumether (1:4) gave 5-N,N-bis(2-chloroethyl)amino!-2,4-dinitrothiobenzamide (13) Ic: X=Cl,R=CSNH₂) (0.48 g, 92%), mp (EtOAc/petroleum ether) 166°-167° C. ¹ H NMR(CD₃ SOCD₃) δ9.50, 9.34 (2xbr, 2 H, CSNH₂), 8.50 (s, 1 H, H-3), 7.45 (s,1 H. H-6), 3.87, 3.82 (2xm. S H, NCH₂ CH₂ Cl). ¹³ C NMR δ199.46 (CS),148.08 (C-5), 143.87 (C-1), 139.51. 136.99 (C-2.4), 125.23, 121.36(C-3,6), 54.23 (CH₂ N), 42.02 (CH₂ Cl). Anal. (C₁₁ H₁₂ Cl₂ N₄ O₄ S)C,H,N,S.

EXAMPLE D Preparation of 5-N,N-bis(2-chloroethyl)amino!-2,4-dinitrobenzenesulfonamide (18)(Id:X=Cl, R=SO₂ NH2) by the method outlined in Scheme 4.

A solution of 5-chloro-2,4-dinitrobenzenesulfonamide (VIII) Herbert,R.B. and Hollman, R.G. Tetrahedron, 1965, 21, 663-675! (1.55 g, 5.5mmol) and diethanolarnine (1.16 g, 11 mmol) in dioxan (100 mL) was heldat 60 ° C. for 1.5 h. then adsorbed directly onto silica gel. Excesssolvent was evaporated under reduced pressure, and the residue waschromatographed on silica gel, eluting with EtOAc/MeOH (19:1), to give5- N,N-bis(2-hydroxyethyl)amino!-2,-dinitrobenzenesulfonamide (IX) (1.91g, 99%), mp (EtOAc/petroleum ether) 138°-139° C. ¹ H NMR ((CD₃)₂ SO)δ8.53 (s, 1 H. H-3), 7.97 (s, 1 H. H-6), 7.93 (br s. 2 H, SO₂ NH₂), 4.88(t,J=5.1 Hz, 2 H, OH), 3.65 (dt,J=5.2, 5.1 Hz, 4 H, CH₂ OH), 3.49 (t,J=5.1 Hz, 4 H, NCH₂). ¹³ C NMR δ146.84 (C-5), 140.53 (C-1), 136.84,134.39 (C-2,4), 125.70 (C-3), 54.27 (NCH₃). Anal. (C₁₀ H₁₄ N₄ O₈ S)C,H,N.

A stirred solution of the above diol (IX) (1.90 g, 5.43 mmol) and Et₃ N(1.90 mL. 14 mmol) in dry THF (60 mL) was treated dropwise at 0° C. withmethanesulfonyl chloride (0.89 mL, 11 mmol). After a further 15 min, thesolution was diluted with EtOAc, washed well with water, and worked upto give the crude dimesylate (Id: X=OSO₂,Me, R=SO₂ NH₂). This wasimmediately dissolved in DMF (50 mL) containing LiCl (20 g), and themixture stirred at 130° C. for 15 min before solvent was removed underreduced pressure. The residue was partitioned between EtOAc and water,and the organic layer was worked up and chromatographed on silica gel.Elution with EtOAc gave 5-N,N-bis(2-chloroethyl)amino!-2,4-dinitrobenzenesulfonamide (18) (Id:X=Cl, R=SO₂ NH₂) (1.47 g, 64%), mp 153°-154° C. (EtOAc/petroleum ether).¹ H NMR ((CD₃)₂ CO) δ8.54. (S, 1H, H-3), 7.99 (br, 2H, SO₂ NH₂), 7.91(S, 1H, H-6), 3.84 (t, J=5.6 Hz, 4H, CH₂ Cl), 3.67 (t, J=5.6 Hz, 4H, CH₂N). ¹³ C NMR ((CD₃)₂ CO) δ146.13 (C-5), 140.64 (C-1), 138.86, 136.93(C-2,4), 124.95, 121.77 (C-3,6), 52.47 (CH₂ N), 41.62 (CH₂ Cl). Anal.(C₁₀ H₁₂ Cl₂ N₄ O₆ S) C,H,N,S.

EXAMPLE E Preparation of 2-N,N-bis(2-chloroethyl)amino!-3,5-dinitrobenzamide (19) (Ie: R=CONH₂) andN-(N,N-dimethylaminoethyl) 2-N,N-bis(2-chloroethyl)amino!-3,5-dinitrobenzamide (20) (Ie: R=CONHCH₂CH₂ NMe₂) by the method outlined in Scheme 5.

Reaction of commercially-available 2-chloro-3,5-dinitrobenzoic acid (X)with SOCl₂ gave 2-chloro-3,5-dinitrobenzoyl chloride (XI: Z=Cl), whichwas quenched with ammonia to give 2-chloro-3,5-dinitrobenzamide (XI:Z=NH₂). A solution of this amide (1.00 g, 4.07 mmol) and Et₃ N (1.42 g,10 mmol) in ρ-dioxane (30 mL) was 0356 treated withN,N-bis(2-chloroethyl)amine hydrochloride (1.45 g, 8.14 mmol) at 50° C.for 18 h. The mixture was then poured into water and extracted withEtOAc to give an oil, which was chromatographed on silica gel. Elutionwith EtOAc/petroleum ether (1:1) gave 2-N,N-bis(2-chloroethyl)amino!-3,5-dinitrobenzramide (19) (Ie: R=CONH₂)(1.15 g, 80%), mp (CHCl₃ /petroleum ether) 123°-124° C. ¹ H NMR (CD₃SOCD₃) δ8.70 (d, J=2.7 Hz, 1 H, H-4), 8.49 (d, J=2.7 Hz, 1 H, H-6),7.68, 7.35 (2xbr, 2H, CONH₂), 3.80, 3.61 (2.xt, J=6.9 Hz, y 8H. NCH₂ CU₂Cl). 13C NMR δ167.94 (CONH₂), 147.27 (C-2), 147.2 0. 142.84 (C-3,5),137.70 (C-1), 128.40, 123.24 (C-4:6), 55.84 (CH₂ N), 42.28 ) (CH₂ CI).Anal. (C₁₁ H₁₂ Cl₂,N₄ O₅) C,H,N,Cl.

Similar treatment of the acid chloride (XI: Z=Cl) withN,N-dimethylaminoethyl in water at 0° C. gave N-(N,N-dimethylaminoethyl2-chloro-3,5-dinitrobenzamide (XI, Z=NTHCH₂ CH₂ NMe₂) as a viscous oil(5.6 g. 59%. The hydrochloride salt crystallised from MeOH/isopropylether, mp 220°-222° C. ¹ H NMR (D₂ O) δ8.99 (d, J=2.3 Hz, 1H, ArH4),8.74 (d, J=2.3 Hz. 1H, ArH6), 3.89 (t, J=6.3 Hz, 2H, CH₂ N⁺ Me₂), 3.51(t, J=6.3 Hz, 2H, CONHCH₂), 3.04 (s, 6H, NMe₂). ¹³ C NMR 169.32 (CONH),150.88 (C-2), 148.70, 141.02 (C-3,5), 133.23 (C-1), 129.45, 125.27(C-4,6), 58.64 (CH₂ N^(+Me) ₂), 45.88 (N⁺ Me₂), 37.94 (CONHCH₂). Anal.(C₁₁ H₁₃ ClN₄ O₅.HCl) C,H,N,Cl.

Reaction of this amide (XI: Z=NHCH₂ CH₂ CH₂ NMe₂) withbis(2-chloroethyl)amine hydrochloride and Et₃ N as above, followed bychromatography on silica gel and elution with EtOAc/MeOH (9:1) gaveN-(N,N-dimethylaminoethyl) 2-N,N-bis(2-chloroethyl)amino!-3,5-dinitrobenzamide (20) (Ie: R=CONHCH₂CH₂ NMe₂), mp (EtOAc/petroleum ether) 115°-118° C. ¹ H NMR (CDCl₃) δ8.63(s, 2H, ArH4), 7.41 (br, 1H, CONH), 3.74 (t, J=6.3 Hz, 4H, CH₂ Cl), 3.59(t, J=5.8 Hz, 2H, CONHCH₂), 3.55 (t, J=6.3 Hz, 4H, NCH₂ CH₂ Cl), 2.56(t, J=5.8 Hz, 2H, CH₂ NMe₂), 2.27 (s, 6H, NMe₂). ¹³ C NMR 164.70 (CO),145.93 (C-2), 144.86. 141.94 (C-3,5), 135.68 (C-1), 129.63 (C4), 123.16(C-6), 57.51 (CONHCH₂), 54.82 (NCH₂ CH₂ Cl), 45.12 (NMe₂), 41.39(NCH_(2CH) ₂ Cl), 37.67 (CONHCH₂). Anal. (C₁₅ H₂₁ Cl₂ N₅ O₅) C,H,N,Cl.(The hydrochloride salt was too hygroscopic to handle).

EXAMPLE F Preparation of 3-N,N-bis(2-chloroethyl)amino!-2,6-dinitrobenzamide (21) (If: R=CONH₂) andN-(N,N-dimethylaminoethyl) 3-N,N-bis-(2-chloroethyl)amino!-2,6-dinitrobenzamide (22) (If: R=CONHCH₂CH2NMe₂) by the method outlined in Scheme 6.

A solution of 3-chlorobenzoic acid (60 g, 0.38 mol) in c.H₂ SO₄ (600 ml)was treated portionwise with fuming nitric acid (d 1.42) (150 ml), andthe solution was warmed gradually with stirring to 140° C. in an openflask, and held at this temperature for 6h. After cooling overnight,ice-water was added cautiously, and after chilling to 5° C. for 3h theprecipitated product was removed by filtration and washed well withwater. Crystallisation from aqueous EtOH gave5-chloro-2,4-dinitrobenzoic acid (II: Y=OH) (62 g, 66%) mp 180°-183° C.Goldstein, H.; Stamm, R. Helv. Chim. Acta, 1952, 35, 1330-1332 report mp182°-183° C.!. The original filtrate and washings were combined andallowed to stand for several hours, depositing crystals of pure3-chloro-2,6-dinitrobenzoic acid (XII) (8.4 g, 9%), mp (H₂ O)162°-163.5° C. ¹ H NMR (CD₃ SOCD₃) δ10.50 (br, 1 H, COOH), 8.82 (d,J=8.8 Hz, 1 H, H-5), 8.62 (d, J=8.8 Hz, 1 H, H4). ¹³ C NMR δ162.03(COOH), 146.34 (C-2), 145.57 (C-6), 133.69 (C-5), 130.01 (C-1), 128.26(C4), 125.19 (C-3). Anal. (C₇ H₃ ClN₂ O₆) C,H,N,Cl.

The 3-chloro-2,6-dinitrobenzoic acid (XI) was converted to the ethylester, which was reacted as above with diethanolamine to give crudeethyl 3- N,N-bis-(2-hydroxyethyl)amino!-2,6-dinitrobenzoate (XIII: X=OH,Z=OEt). Reaction of this with MsCl/LiCl gave ethyl 3-N,N-bis(2-chloroethyl)amino!-2,6-dinitrobenzoate (XII: X=Cl, Z=OEt), mp(EtOAc/petroleum ether) 84°-87° C. ¹ H NMR (CD₃ COCD₃) δ8.32 (d, J=9.4Hz, 1H, H-5), 7.79 (d, J=9.4 Hz, 1H, H4) 4.39 (q, J=7.1 Hz, 2H, OCH₂CH₃), 3.85-3.77 (m, 8H, NCH₂ CH₂ Cl), 1.34 (t, J=7.1 Hz, 3H, OCH₂ CH₃).¹³ C NMR δ163.24 (COOEt), 148.30 (C-3), 141.04, 138.80 (C-2,6), 128.84,124.65 (CA,4.5), 127.39 (C-1), 63.94 (OCH₂ CH₃), 54.39 (NCH₂), 42.00(CH₂ Cl) 13.89 (OCH₂ CH₃). Anal. (C₁₃ H₁₅ Cl₂ N₃ O₆) C,H,N,Cl.

An X-ray crystallographic determination was carried out on this compoundto confirm the structure.

Hydrolysis of this ester (XII: X=Cl, Z=OEt) in aqueous KOH/ρ-dioxane (30mL) at 20° C. for 18h gave 3-N,N-bis(2-chloroethyl)amino!-2,6-dinitrobenzoic acid (XIII: X=Cl, Z=OH),which was converted to the corresponding acid chloride (XIII: X=Cl,Z=Cl). A solution of this in Me₂ CO at 0° C. was treated with excessaqueous ammonia, and the product was chromatographed on silica gel,EtOAc/petroleum ether (1:1) eluting 3-N,N-bis(2-chloroethyl)amino!-2.6-dinitrobenzamide (21) (If: R=CONH₂)(0.93 g), mp (EtOAc/petroleum ether) 140°-141.5° C. ¹ H NMR (CD₃ COCD₃)δ8.26 (d, J=9.3 Hz. 1H, H-5), 7.71 (d, J=9.3 Hz, 1H, H-4), 7.63, 7.23(2xbr, 2H, CONH₂), 3.75 (s, 8H, NCH₂ CH₂ Cl). ¹³ C NMR δ163.99 (CONH₂),147.38 (C-3), 142.92, 139.76 (C-2,6), 130.48 (C-1), 128.22, 124.23(C4,5), 54.74 (NCH₂), 42.00 (CH₂ Cl). Anal. (C₁₁ H₁₂ Cl₂ N₄ 0₅) C,H,N,Cl

Similar treatment of the acid chloride (XIII: X=Cl, Z=Cl) withN,N-dimethylethylenediamine gave N-(N,N-dimethylaminoethyl) 3-N,N-bis(2-chloroethyl)amino!-2,6-dinitrobenzamide (22) (If: R=CONHCH₂CH₂ NMe₂). The hydrochloride salt crystallised from MeOH/isopropylether, mp 180°-182 ° C. 1H NMR (D₂ O) δ8.37 (d, J=9.6 Hz, 1H, ArH5),7.63 (d, J=9.6 Hz, 1H, ArH4), 3.80-3.71 (m, 10H, NCH₂ CH₂ Cl and CH₂NMe₂), 3.44 (t, J=6.4 Hz, 2H, CONHCH₂), 3.00 (s, 6H, NMe₂) ¹³ C NMRδ168.75 (CONH), 150.91 (C-3), 141.55, 138,72 (C-2,6), 131.61 (C-4),130.45 (C-1), 125.85 (C-5), 58.31 (CH₂ N³⁰ Me₂), 55.74 (NCH₂ CH₂ Cl),45.97 (N³⁰ Me₂), 44.16 (NCH2CH₂ Cl ), 37.91 (CONHCH₂). Anal. (C₁₅ H₂₁Cl₂ N5O₅. HCl) C,H,N,Cl.

EXAMPLE G: Preparation of 4-N,N-bis(2-chloroethyl)amino!-3,5-dinitrobenzamide (23) (Ig: X=Cl,R=CONH₂) and N-(N,N-dimethylaminoethyl) 4-N,N-bis(2-chloroethyl)amino!-3,5-dinitrobenzamide (24) (Ig: X=Cl,R=CONHCH₂ CH2NMe₂) by the method outlined in Scheme 7.

A solution of methyl 4-chloro-3,5-dinitrobenzoate (XIV) (10.0 g, 0.036mol) and diethanolamine (7.66 g, 0.073 mol) in ρ-dioxane (60 mL) wasstirred at 50° C. for 5h. Volatiles were removed under reduced pressure,and the residue was adsorbed directly onto silica gel andchromatographed. Elution with EtOAc/petroleum ether (1:10) gaveforeruns, and subsequent elution with EtOAc/petroleum ether (3:2) gavemethyl 4- N,N-bis(2-hydroxyethyl)amino!-3,5-dinitrobenzoate (XV: X=OH,Z=OMe) (9.61 g, 80%), mp (CHCl₃ /petroleum ether) 101°-103° C. ¹ H NMR(CDCl₃) δ8.52 (s, 2H, ArH-2,6), 3.97 (s, 3 H, COOCH₃), 3.76 (dxt, J=6.5,4.7 Hz, 4H, CH₂ OH), 3.26 (t, J=4.7 Hz, 4H, NCH₂), 2.76 (t, J=6.5 Hz, 2H, OH). ¹³ C NMR δ163.14 (COOCH₃), 145.50 (C-4), 142.54 (C-3,5), 131.19(C-2,6), 123.59 (C-1), 59.16 (CH₂ OH)1, 54.58 (CH₂ N), 53.14 (OCH₃).Anal. (Cl₁₂ H₁₅ N₃ O₈) C,H,N.

MsCl (3.93 rnL, 0.049 mol) was added dropwise at 0 ° C. to a solution ofthe above diol (XV: X=OH, Z=OMe) (7.30 g, 0.022 mol) and Et₃ N (7.72 mL,0.055 mol) in CH₂ C₂ Cl₂ (100 mL). After 15 min, the solution was washedseveral times with water and worked up to give the crude dimesylate,which was treated with LiCl in DMF at 120 ° C. for 5 min. Workup andchromatography on silica gel (elution with EtOAc/petroleum ether (1:4))gave methyl 4- N,N-bis(2-chloroethyl)amino!-3,5-dinitrobenzoate (XV:X=Cl, Z=OMe) (6.21 g, 77%), mp (CHCl₃ /petroleum ether) 93°-95° C. ¹ NMR(CDCl₃) δ8.54 (s, 2 H. H-2,6), 3.99H (s, 3H, COOCH₃), 3.63 (t, J=6.9 Hz,4H, CH₂ Cl), 3.43 (t, J=6.9 Hz, 4H, CH₂ N). ¹³ C NMR δ162.90 (COOCH₃),147.83 (C4), 140.53 (C-3,5), 129.85 (C-2,6), 126.70 (C-1), 55.47 (CH₂N), 53.23 (OCH₃), 41.18 (CH₂ Cl). Anal. (C₁₂ H₁₃ Cl₂ N₃ O₆) C,H,N,Cl.

Hydrolysis of this ester (XV: X=Cl,Z=OMe) with aqueous KOH/ρ-dioxane at20° C. for 3h gave a quantitative yield of 4-N,N-bis(2-chloroethyl)amino!-3,5-dinitrobenzoic acid (XV: X=Cl, Z=OH),mp EtOAc/petroleum ether) 164°-166° C. ¹ NMR ((CD₃)₂ SO) δ8.61 (s, 2H,H-2,6), 3.78 (t, J=6.9 Hz, 4H, CH₂ Cl), 3.52 (t, J=6.9 Hz, 4H, CH₂ N)(COOH not visible). ¹³ C NMR δ164.12 (COOH), 149.12 (C-4), 141.40(C-3,5), 130.70 (C-2,6), 128.19 (C-1), 56.14 (CH₂ N), 42.46 (CH₂ Cl).Anal (C₁₁ H₁₁ Cl₂ N₃ O₆) C,H,N,Cl.

The above acid (XV: X=Cl, Z=OH) was converted to the crude acid chloride(XV: X=Cl, Z=Cl), which was dissolved in dry Et₂ O (100 mL), cooled to5° C., and treated dropwise with aqueous NH₃ (20 mL of a 3N solution, 60mmol). After 10 min the solution was worked up, and the residue waschromatographed on silica. Elution with EtOAc/petroleum ether (3:7) gaveforeruns, while EtOAc/petroleum ether (7:3) gave 4-N,N-bis(2-chloroethyl)amino!-3,5-dinitrobeazamide (23) (Ig: X=Cl,R=CONH₂) (80% overall yield), mp (EtOAc/petroleum ether) 113°-116 ° C. ¹H NMR (CD₃ COCD₃) δ8.61 (s, 2 H, H-2,6), 7.98, 7.24 (2xbr, 2H, CONH₂),3.75 (t, J=7.0 Hz, 4H, CH₂ Cl), 3.50 (t, 4 H, CH₂ N). ¹³ C NMR δ164.91(CONH₂), 149.32 (C-4), 140.07 (C-3,5), 132.18 (C-1), 128.91 (C-2,6),56.37 (CH₂ N), 42.55 (CH₂ Cl). Anal. (C₁₁ H₁₂ Cl₂ N₄ O₅) C,H,N,Cl.

Similar treatment of the crude acid chloride (XV: X=Cl, Z=Cl) withN,N-dimethylethylenediamine gave N-(N,N-dimethylaminoethyl) 4-N,N-bis(2-chloroethyl)amino!-3,5-dinitrobenzamide (24) (Ig: X=Cl,R=CONHCH₂ CH₂ NMe₂). The hydrochloride salt crystallised fromMeOH/isopropyl ether, mp 145°-148° C. ¹ NMR (CD₃ SOCD₃) δ10.65 (br, 1H,HCl), 9.49 (t, J=5.4 Hz, 1H, CONH), 8.75 (s, 2H, ArH2,6), 3.71 (t, J=6.9Hz, 4H, NCH₂ CH₂ Cl ), 3.44-3.28 (m, 8H, NCH₂ CH₂ Cl and CH₂ CH₂ N⁺Me₂), 2.83 (s, 6H, N⁺ Me₂). ¹³ C NMR δ162.63 (CO), 147.43 (C-3,5),138.75 (C-4), 130.22 (C-1). 128.31 (C-2,6), 55.44 (CH₂ N⁺ Me₂), 54.75(NCH₂ CH₂ Cl), 42.22 (N⁺ Me₂), 42.18 (NCH₂ CH₂ Cl), 34.75 (CONHCH₂).Anal. (C₁₅ H₂₁ Cl₂ N₅ O₅.HCl) C,H,N,Cl.

The following Table 2 gives biological data for selected examples of thecompounds listed in Table 1. The abbreviations used in Table 2 are:

No.

The number given the corresponding compound in Table 1.

IC₅₀

Growth inhibition studies were performed as described in detailelsewhere (W.R. Wilson, R.F. Anderson and W.A Denny. J Med. Chem., 1989,32, 23; G.J. Finlay, B.C. Baguley and W.R. Wilson. Anal Biochem., 1984,139, 172.), using 200 viable AA8 or 300 viable UV4 cells plus 5000lethally-irradiated AA8 feeder cells per well in 96-well tissue culturedishes. AA8 and UV4 cells were maintained in logarithmic-phase growth in25 cm³ tissue culture flasks with subculture twice weekly bytrypsinization. The growth medium was antibiotic-free α-MEM with 10% v/vheat-inactivated (560° C., 40 min) fetal calf serum. Doubling times wereapproximately 14h for AA8 and 15h for UV4 cells. Cultures were testedfor mycoplasma contamination frequently, using, a cytochemical stainingmethod (I.R. Chen, Exp. Cell Res., 1977, 104, 255. Drugs were added 24hours after initiating cultures in 96-well dishes, and cells wereincubated under aerobic or hypoxic conditions for 18 hours beforewashing with fresh medium. The IC₅₀ was determined as the drugconcentration needed to reduce the cell mass (protein content, measuredafter 72-78h by staining with methylene blue and measuring absorbance ina microplate photometer) to 50% of the mean value for 8 control cultureson the same 96-well plate.

HF_(air)

Ratio of IC₅₀ values for a compound after exposure as detailed aboveagainst AA8 and UV4 cell lines (HF=IC₅₀ (AA8)/IC₅₀ (UV4)).

CT₁₀

The product (in μM-hr) of the drug concentration times the exposure timeto reduce cell survival to 10% of control values in the clonogenicassay.

air/N₂

Ratio of CT₁₀ values after exposure of UV4 cells as detailed above undereither aerobic (air) or hypoxic (N₂) conditions (=aerobic CT₁₀ /hypoxicCT₁₀).

HF_(NR-2)

Ratio of IC₅₀ values for a compound after exposure as detailed aboveagainst the UV4 cell line in the presence and absence of the E. colinitroreductase enzyme NTR-2.

                  TABLE 2                                                         ______________________________________                                        Biological activity of selected compounds of Table I                                                              IC.sub.50                                                                            HF                                     IC.sub.50 (AA8)                                                                         HF     CT.sub.10 (UV4)                                                                       CT.sub.10 ratio                                                                      (UV4)  (NR-2)                             No  (air: μM)                                                                            (air)  (air: μM-h)                                                                        (air/N.sub.2)                                                                        (air: μM)                                                                         (air)                              ______________________________________                                         1  468       6.8    2925    58     109    73.5                                2  57        7.5    160     20     10.9   32.3                                3  34        2.6    170     42     15     73.8                                4  >200      --     1200    17     99     10.0                                6  10600     1.5    >30000  >2     317    0.76                                7  24        6.8    86      8.5    2.3    9.2                                 8  >100      --     >360    --                                                9  310       1.4    >3500   >20                                              10  264       23     156     1.6                                              11  230       4.2    1130    60                                               12  920       3.0    3700    45                                               13  >25       >2     >60     >3                                               14  7300      1.7    <14400  <10                                              15  6100      1.1    14000   2                                                17  1790      3.2    3340    14                                               18  3         --     --      --                                               19  >100      ca. 3  >300    >5                                               20  150       14     1200    36                                               21  >240      >2     >300    >3                                               22  426       3.2    3600    130                                              23  97        1.4    >450    >1                                               24  62        2.0    >273    >1                                               ______________________________________                                    

The following Examples 1 to 3 show that compound 1 (referred to thereinas SN23862) is reductively activated by bacterial nitroreductaseenzymes.

The alkylating agent 5-(aziridin-1-yl)-2,4-dinitrobenzaride (hereinafterdesignated CB 1954) has been known, almost for 20 years, as aninteresting experimental compound of unique selectivity. Although CB1954 is structurally quite closely related to numerous other knownalkylating agents which have a relatively broad range of activity, CB1954 exhibits considerable activity against the Walker tumour cells invivo or in vitro but was thought to be virtually inactive against othertumours.

It was recently discovered that the selectivity of CB 1954 arose fromthe fact that it was not an anti-tumour agent per se but was a prodrugfor an anti-tumour agent generated from CB 1954 by a nitroreductaseenzyme found in the Walker cell. This nitroreductase from the Walkercell was subsequently shown to be an enzyme known from other sourceswhich was an AND(P)H dehydrogenase (quinone) classified as EC.1.6.99.2,see Robertson et al, J. Biol. Chem. 261, 15794-15799 (1986).

In the course of the previous investigations with CB 1954,it was foundthat the Walker cell enzyme EC.1.6.99.2 had the ability to reduce the4-nitro group of CB 1954 to the corresponding hydroylamine and that itwas the resulting 5-(aziridin-1-yl)-2-nitro-4-hydroxylamino-benzamidethat was the active anti-tumour agent.

PCT Application PCT/GB92/01947 of Cancer Research Campaign TechnologyLimited describes new nitroreductases, obtainable from bacterialsources, for example from Escherichia coli and from Bacillusamyloliquifaciens, that are of interest in that not only are theycapable of converting CB 1954 into an active anti-tumour agents butalso, unlike EC.1.6.99.2. capable of converting CB 1954 analogues whichare also prodrugs into active anti-tumour agents.

As is more particularly described in PCT Application PCT/GB92/01947 theuse of prodrugs represents a clinically very valuable concept in cancertherapy since, particularly where the prodrug is to be converted to ananti-tumour agent under the influence of an enzyme that is linkable to amonoclonal antibody that will bind to a tumour associated antigen, thecombination of such a prodrug with such an enzyme monoclonal/antibodyconjugate represents a very powerful clinical agent.

EXAMPLE 1

The effect of SN23862 on the survival of V79 cells in the presence ofthe E. coli nitroreductase. all treatments were for 2 hours at 37° C.and the cells were then plated out for their resulting colony formingability. The nitroreductase concentration was 2 μg/ml and NADH was usedas a co-factor. The initial cell density was 2×10⁵ /mL.

    ______________________________________                                        TREATMENT     % SURVIVAL  % DRUG REDUCTION                                    ______________________________________                                        CONTROL       100         --                                                  +500 μM NADH                                                                             100         --                                                  +50 μM SN23862                                                                           100         <1.0                                                +NADH + SN23862                                                                             100         <1.0                                                +Nitroreductase                                                                             94          --                                                  +NR + 50 μM SN23862                                                                      99          <1.0                                                +NR + SN23862 + 500 μM                                                                   0.007       95                                                  NADH                                                                          ______________________________________                                    

EXAMPLE 2

The effect of SN23862 on the survival of V79 cells in the preence of theB. amyloliquifaciens nitroreductase. All treatments were for 2 hours at37° C. and the cells were then plated out for their resulting colonyforming ability. The nitroreductase concentration was 7.45 μg/ml andNADII was ised as a co-factor. The initial cell density was 2×10⁵ /mL.

    ______________________________________                                        TREATMENT     % SURVIVAL  % DRUG REDUCTION                                    ______________________________________                                        CONTROL       100         --                                                  +500 μM NADH                                                                             120         --                                                  +50 μM SN23862                                                                           110         <1.0                                                +NADH + SN23862                                                                             115         <1.0                                                +Nitroreductase (BA)                                                                        96.8        --                                                  +BA + 50 μM SN23862                                                                      110         <1.0                                                +BA + SN23862 + 500 μM                                                                   6.0         >95                                                 NADH                                                                          ______________________________________                                    

EXAMPLE 3

The effect of SN23862 on the survival of V79 or Mawi cells in thepresence of the E. coli nitroreductase: A5B7 F(ab)₂ conjugate. Alltreatments were for 2 hours at 37° C. and the cells were then plated outfor their resulting colony forming ability. The conjugate concentrationwas 81 μg/ml (euivalent to 5.2 μg/ml nitroreductase) and NADII was usedas a co-factor. The initial cell density was 2×10⁵ /mL. No attempt wasmade to remove unbound conjugate.

    ______________________________________                                                 % SURVIVAL                                                           TREATMENT  MAWI Cells                                                                              V79 Cells                                                                              % DRUG REDUCTION                                ______________________________________                                        CONTROL    100       100      --                                              +500 μM NADH                                                                          104.6     94.6     --                                              +50 μM SN23862                                                                        90.9      94.3     <1.0                                            +NADH + SN23862                                                                          93.1      80.4     <1.0                                            +Conjugate (CG)                                                                          85.5      85.8     --                                              +CG + 50 μM                                                                           94.6      73.5     <1.0                                            SN23862                                                                       +CG + SN23862 +                                                                          0.014     0.003    >99                                             500 μM NADH                                                                ______________________________________                                    

The accompanying FIG. 1 shows radiosensitising properties of compound 1.

The radiosensitising efficiency of compound 1 has been assessed instirred suspensions of AA8 cells under hypoxie conditions using gammairradiation (cobalt 60) at a dose rate of 2.2 Gy/min at 37° C. Drug andcells were mixed under hypoxic conditions 30 min before irradiation.Radiosensitisation of hypoxic cells was observed (FIG. 1). Theconcentration for an enhancement ratio (ratio of radiation doses for 10%survival with and without drug) of 1.6 (C₁.6) was approximately 100 μM,and this extent of sensitisation was observed in the absence of drugtoxicity (FIG. 1). Compound 1 is therefore a hypoxic cellradiosensitiser as well as a hypoxia-selective cytotoxin.

It is clear from the data of Table 2, Examples 1 to 3 and FIG. 1 thatthe examples of the nitroaniline derivatives of general formula (I)listed in Table 2 include compounds which are active as cytotoxicagents, and which have the additional capability of being reductivelyactivated by both mammalian tumour cells and bacterial nitroreductaseenzymes, and are therefore suitable as prodrugs. The compounds areselectively toxic to hypoxic tumour cells, and therefore useful asanticancer drugs. The compounds are capable of sensitising hypoxic cellsin vitro to ionising radiation and thus are suitable as hypoxic cellradiosensitisers.

The present invention therefore also provides pharmaceuticalcompositions having antitumour and radiosensitising activity andcomprising at least one compound represented by the general formula (I),and one or more pharmaceutically-acceptable carriers or diluents.

The present invention further provides the use of at least one compoundrepresented bv the general formula (I) for the manufacture ofpharmaceutical compositions for the treatment of tumours, in particularcancers.

When the compounds of formula (I) are used as reductively-activatedprodrugs for cytotoxins, they can be used in association withnitroreductase enzymes, including the nitroreductases described inApplication PCT/GB92/01947, and so provide a system of cancerchemotherapy where the extent of exposure of the patient to thecytotoxic agent is limited, so far as possible, to those regions wherethere is the inter-reaction between the prodrug and the nitroreductase.

The most or one of the most convenient ways of utilising such a systemof cancer chemotherapy is to conjugate the nitroreductase to atargetting agent such as a monoclonal antibody that will bind with atumour-associated antigen.

The term "monoclonal antibody" will be understood by those of skill inthe art not simply to refer to antibodies produced by traditionalhybridoma techniques, but also to cover antibodies and variants thereofproduced by recombinant means. These include, for example, humanisedantibodies such as those with a constant region from a human antibodygrafted onto a non-human antibody variable region (see for exampleEP-A-0 120 694), chimeric antibodies such as those with non-humancomplementarity determining regions (CDRs) grafted into a human variableregion framework (see for example EP-A-0 239 400) and single, chainantibodies. Fragments of such monoclonal antibodies which retain theirtarget binding activity are also included by the general term"monoclonal antibody". This includes Fab' and F(ab')₂ fragments.

The selection of monoclonal antibody will clearly be influenced by thenature of the target tumour but for the purposes of illustration,reference may be made to the anti-CEA antibody A₅ B₇.

With this system, it is possible in a course of cancer chemotherapy toadminister to the patient requiring the treatment the nitroanilinecompound which is the prodrug for a cytotoxic compound and theenzyme/targeting agent conjugate. The prodrug and the conjugate can beadministered simultaneously but it is often found preferable, inclinical practice, to administer the enzyme/agent conjugate before theprodrug; e.g. up to 72 hours before, in order to give the enzyme/agentconjugate an opportunity to localise in the region of the tumour target.By operating in this way, when the prodrug is administered, conversionof the prodrug to the cyrotoxic agent tends to be confined to theregions where the enzmye/agent conjugate is localised, i.e. the regionof the target tumour and damage to healthy cells caused by the prematurerelease of the cytotoxic agent is minimised.

The degree of localisation of the enzyme/agent conjugate (in terms ofthe ratio of localized to freely circulating active conjugate) can befurther enhanced using the clearance and/or inactivation systemsdescribed in WO89/10140. This involves, usually following administrationof the conjugate and before administration of the prodrug, theadministration of a component (a "second component") which is able tobind to the such part of the conjugate so as to inactivate the enzymeand/or accelerate the clearance of the conjugate from the blood. Such acomponent may include an antibody to the nitroreductase which is capableof inactivating the enzyme.

The second component may be linked to a macromolecule such as dextran, aliposome, albumin, macroglobulin or a blood group O erythrocyte so thatthe second component is restrained from leaving the vascularcompartment. In addition or as an alternative, the second component mayinclude a sufficient number of covalently bound galactose residues, orresidues of other sugars such as lactose or mannose, so that it can bindthe conjugate in plasma but be removed together with the conjugate fromplasma by receptors for galactose or other sugars in the liver. Thesecond component should be administered and designed for use such thatit will not, to any appreciable extent, enter the extravascular space ofthe tumour where it could inactivate localised conjugate prior to andduring administration of the prodrug.

The exact dosage regime will, of course, need to be determined byindividual clinicians for individual patients and this, in turn, will becontrolled by the exact nature of the prodrug and the cytotoxic agent tobe released from the prodrug but some general guidance can be given.Chemotherapy of this type will normally involve parenteraladministration of both the prodrug and the enzyme/agent conjugate andadministration by the intravenous route is frequently found to be themost practical.

The active compounds may be orally administered, for example, with aninert diluent or with an assimilable edible carrier, or they may beenclosed in hard or soft shell gelatin capsules, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompounds may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets troches, capsules, elixirs,suspensions, syrups wafers and the like. Such compositions andpreparations should contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 and about 60% of theweight of the unit. The amount of active compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained. Preferred compositions or preparations according to thepresent invention are prepared so that an oral dosage unit form containsbetween about 5 and about 200 meg of active compound.

The tablets, troches, pills, capsules and the like may also contain tilefollowing: a binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin may be added or a flavouring agent such aspeppermint, oil of wintergreen or cherry flavouring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills or capsules may be coated with shellac,sugar or both. A syrup or elixir may contain the active compound,sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavouring such as cherry or orange flavour. Ofcourse any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparations and formulations.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compound as a free base orpharmaceutically acceptable salt can be prepared in water suitably mixedwith a surfactant such as hydroxypropylcellulose. Dispersions can alsobe prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisns.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability, exists. It must be stable under theconditions of manufacture and storage and must be preserved against becontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, elycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof andvegetable oils. The proper fluidity can be maintained, for example; bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersions and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about bv various antibacterial and antifungal agents, forexample, parabens, clorobutanol, phenol, sorbic acid, thimerosal and thelike. In many cases, it will be preferable to include isotonic agents,for example, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminiummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, "pharmaceutically acceptable carrier" includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuitable as unitary dosages for the mammalian subjects to be treated;each unit containing a predetermined therapeutic effect in associationwith the required pharmaceutical carrier. The specification for thenovel dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active material andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active materialfor the treatment of disease in living subjects having a diseasedcondition in which bodily health is impaired.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically-acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit dosage form can, for example, contain the principalactive compound in amounts ranging from about 0.1 to about 400 mg, withfrom about 1 to about 30 mg being preferred. Expressed in proportions,the active compound is generally present, in from about 0.1 to 400 mg/mlof carrier. In the case of compositions containing supplementary activeingredients, the dosages are determined bv reference to the usual doseand manner of administration of the said ingredients.

What we claim is:
 1. A compound represented by the formula (I), ##STR4##where NO₂ is in the 2-position, R represents CONHCH₂ CH₂ NMe₂, Arepresents 4-NO₂ and B represents 5-N(CH₂ CH₂ Cl)₂.
 2. A compoundrepresented by the formula (I), ##STR5## where NO₂ is in the 2-position,R represents CONHCH₂ CH₂ N()Me₂, A represents 4-NO₂ and B represents5-N(CH₂ CH₂ Cl)₂.
 3. A compound represented by the formula (I), ##STR6##where NO₂ is in the 2-position, R represents CONHCH₂ CH₂ NMe₂, Arepresents 6-NO₂ and B represents 5-N(CH₂ CH₂ Cl)₂.
 4. A compoundrepresented by the formula (I), ##STR7## where NO₂ is in the 3-position,R represents CONHCH₂ CH₂ NMe₂, A represents 5-NO₂ B represents 2-N(CH₂CH₂ Cl)₂.